Synapses are fundamental to nervous system function and information processing in the normal and pathological brain. They are highly dynamic structures capable of supporting high firing rates and displaying a broad range of plasticity. Our long-term goal is to determine how the dynamic recruitment and loss of specific presynaptic proteins govern the ongoing pattern of synaptic transmission and plasticity. A detailed picture of the molecular interactions occurring within a synapse is required to understand how synaptic protein dynamics ultimately shape activity in the nervous system in health and disease. In this proposal, we will investigate complexin, a highly conserved molecule that is crucial for proper synaptic function. Mice lacking both major isoforms of complexin die at birth, and loss of a single isoform is associated with profound locomotor, sensory, and behavioral deficits. Complexin expression is altered in a host of psychiatric and neurodegenerative diseases including Huntington's, Parkinson's, and Alzheimer's disease. Although it is well-established that complexin plays a major role in synaptic function, its mode of action is controversial: there is evidence for both facilitatory and inhibitory roles of this protein in the regulation of synaptic transmission. We propose to characterize the function and mechanism of complexin action at the synapse in C. elegans using a combination of electrophysiology, genetics, and in vivo dynamic imaging. We have developed innovative methods of investigating protein interactions in vivo by monitoring the dynamics of proteins exchanging between neighboring synapses using photoactivatable GFP. We will deploy these methods in C. elegans as a model system in which to study the protein interactions of complexin and its binding partners in a functional synapse. By mutating either complexin or its binding partners, we have established that affinity changes lead to mobility changes. We have also identified novel interactions that recruit complexin to the synapse. We will elucidate the structure of the complexin protein domain that mediates this interaction using a combination of biochemical and spectroscopic techniques. The experiments proposed here should provide new insights into the mechanism of CPX action at the synapse and ascertain to what extent SNARE interactions account for the recruitment of this essential protein. We anticipate that the study of in vivo protein interactions in the context of synaptic function will be successfully extended to include many other synaptic proteins. The biophysical and molecular details of these dynamics interactions will greatly enrich our understanding of the synapse. PUBLIC HEALTH RELEVANCE: Synaptic connections are the fundamental building blocks of the brain. Many psychiatric and neurodegenerative diseases involve pathological synaptic function at the cellular and molecular level, but little is known about how synaptic molecules operate in health and in disease. Using a powerful combination of genetics, live animal imaging, and physiology, we propose to study complexin, an essential protein required for proper synapse function.